Composition for analyzing nucleic acid
a nucleic acid and nucleic acid technology, applied in the field of nucleic acid composition, can solve the problems of reducing the analysis sensitivity, emitted background light, and extremely expensive datps in comparison with datp, and achieve the effect of suppressing the emission of background ligh
Active Publication Date: 2010-10-07
KIKKOMAN CORP
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AI Technical Summary
Benefits of technology
[0017]According to the present invention, highly sensitive analyses of nucleic acids can be carried out while suppressing the effects of emission of background light attributable to dATP.
Problems solved by technology
Thus, when analyzing nucleic acid in a reaction liquid containing this dATP using a luciferin-luciferase reaction, the presence of the dATP increases the amount of background light emitted, resulting in the problem of a decrease in analysis sensitivity.
However, dATPαS is extremely expensive in comparison with dATP, and has problems such as poor incorporation efficiency into DNA polymerase reactions and preventing the complementary chain synthesis reaction from proceeding properly.
However, when the rate of content of dATP of dATP is reduced, the complementary chain synthesis is unable to be fully completed due to a shortage of the required amount of dATP in the case where thymine (T) is continued in the sequence of the template DNA.
Thus, the amount of emitted signal light does not correspond to the number of thymine, and since unreacted thymine remains, the reaction is unable to proceed to the next sequence resulting in the problem of being unable to accurately determine the base sequence being analyzed.
Method used
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Effect test
example 1
[0052]Photinus pyralis luciferase available from Sigma was used for the Photinus pyralis luciferase (wild type). In addition, LUC-H (Kikkoman, product code: 61314) refers to a luciferase having improved stability by altering the amino acid at position 217 of Luciola lateralis to leucine (L) and that at position 490 to lysine (K), while LUC-T (Kikkoman, product code: 61315) refers to a luciferase having improved stability by altering the amino acid at position 217 of Luciola cruciata to isoleucine (I). Moreover, LUC-C (Kikkoman, product code: 61313) refers to luciferase having the amino acid sequence of positions 1 to 448 of Luciola cruciata on the side of N-terminal thereof, each of the amino acids at positions 217, 219 and 239 being altered to isoleucine (I), and having the amino acid sequence at positions 447 to 550 of Photinus pyralis luciferase on the side of C-terminal thereof.
Preparation of pET16b-BLU-Y
[0053]A primer for full-length (SEQ ID NO. 2) was synthesi...
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Abstract
The present invention provides a firefly luciferase for inexpensive, highly accurate and highly sensitive nucleic acid analysis that uses dATP instead of an expensive reagent having low reactivity to DNA polymerase in the manner of dATPαS, a method of analyzing nucleic acid that uses that luciferase, and a kit for analyzing nucleic acid thereof.The present invention relates to a composition for analyzing nucleic acid that contains luciferase for which reactivity to dATP is equal to or less than 1 / 400 reactivity to ATP, a method of analyzing nucleic acid that comprises the use of that composition, and a kit for analyzing nucleic acid comprising that composition.
Description
TECHNICAL FIELD[0001]The present invention relates to a composition for analyzing nucleic acid that contains a mutant firefly luciferase, a method of analyzing nucleic acid using thereof, and a kit for analyzing nucleic acid.BACKGROUND ART[0002]Firefly luciferase is an enzyme that emits light by converting ATP, D-luciferin and oxygen to AMP, oxyluciferin and carbon dioxide.[0003]Currently known examples of nucleic acid analysis methods based on detection of emitted light using firefly luciferase include a pyrosequencing method (see, for example, Non-Patent Document 1), a gene polymorphism (SNPs) analysis method using the BAMPER method (see, for example, Non-Patent Document 2), and a gene detection method using a hybridization method (see, for example, Non-Patent Document 3).[0004]These nucleic acid analysis methods are based on the principle of converting pyrophosphoric acid, which is released when a nucleotide is incorporated by complementary chain synthesis, to ATP using an enzyme...
Claims
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Login to View More IPC IPC(8): C12Q1/68C12N9/02
CPCC12Q1/66
Inventor SUZUKI, SHIGEYAKODAMA, YUKAKOKUROSAWA, KEIKOKOBATAKE, EIRYMIE, MASAYASU
Owner KIKKOMAN CORP